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Journal of Biochemistry Advance Access published online on January 23, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn007
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© 2008 The Japanese Biochemical Society

Role of disulfide bonds in a thermophilic serine protease Aqualysin I from Thermus aquaticus YT-1

Masayoshi Sakaguchi, Makoto Takezawa, Rie Nakazawa, Kazutaka Nozawa, Taro Kusakawa, Takeshi Nagasawa, Yasusato Sugahara and Masao Kawakita*

Department of Applied Chemistry, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo 192-0015, Japan

*To whom correspondence should be addressed. Tel: +81-42-628-4857; Fax: +81-42-628-5647; E-mail: bt13004{at}ns.kogakuin.ac.jp

Received October 30, 2007; Accepted January 17, 2008


  Abstract

A thermophilic serine protease, Aqualysin I, from Thermus aquaticus YT-1 has two disulfide bonds, which are also found in a psychrophilic serine protease from Vibrio.sp. PA-44 and a proteinase K-like enzyme from Serratia sp. at corresponding positions. To understand the significance of these disulfide bonds in aqualysin I, we prepared mutants C99S, C194S, and C99S/C194S (WSS), in which Cys69-Cys99, Cys163-Cys194, and both of these disulfide bonds, respectively, were disrupted by replacing Cys residues with Ser residues. All mutants were expressed stably in E. coli. The C99S mutant was 68% as active as the wild-type enzyme at 40°C in terms of kcat value, while C194S and WSS were only 6 and 3%, respectively, as active, indicating that disulfide bond Cys163-Cys194 is critically important for maintaining proper catalytic site conformation. Mutants C194S and WSS were less thermostable than wild-type enzyme, with a half-life at 90°C of 10 min as compared to 45 min of the latter and with transition temperatures on differential scanning calorimetry of 86.7°C and 86.9°C, respectively. Mutant C99S was almost as stable as the wild-type aqualysin I. These results indicate that the disulfide bond Cys163-Cys194 is more important for catalytic activity and conformational stability of aqualysin I than Cys67-Cys99.

Key Words: disulfide bond, serine protease, subtilase, thermostability, activity


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